Our long-term research goal is to understand how structure is coordinated with function in the apical aspect of polarized epithelial cells. We initially identified ezrin, the prototypic member of the ERM (ezrin/radixin/moesin) family, which is a regulated membrane cytoskeletal linking protein of apical microvilli. Activation of ezrin, which involves dissociation of an extended intra-molecular interaction, has been found to unmask binding sites for F-actin and membrane associated proteins, including EBP50, a protein that binds ezrin through a carboxyl-terminal sequence. EBP50 also has two PDZ domains that bind many proteins, including EPI64, a microvillar protein that contains a TBC/RabGAP domain, and nadrin/RICH1, a RhoGAP-containing protein. Our hypothesis is that ezrin has both a membranecytoskeletal linking function and provides a localization site for EBP50 and EPI64, and probably nadrin/RICH1, that are involved in regulating specific membrane traffic between the plasma membrane and endosomes. The goal of the present application is to investigate how ezrin participates in cytoskeletal structure and membrane traffic through EBP50, EPI64 and nadrin/RICH1. First, we shall make structure based site-directed mutations in ezrin that have the potential to activate and/or interfere with specific binding partners, and characterize the properties of the resulting proteins in vitro and in vivo. Second, we shall use biochemical approaches to search among proteins of intestinal and placental microvilli for physiologically relevant new membrane ligands of the PDZ-domains of EBP50. Third, we shall characterize EPI64, especially with respect to its putative RabGAP activity, and explore whether nadrin/RICH1 is a physiologically relevant EBP50 ligand. Fourth, we shall use the information generated in aims 1-3 to study how ezrin, EBP50, EPI64 and nadrin/RICH1 might participate in the endocytic cycle in a general, or specific, manner. This work should provide an integrated approach to the structure and function of the apical aspect of polarized cells. [unreadable] [unreadable]